Antitumor agents

ABSTRACT

The present invention provides illudin analogs of the general formula I:  
                 
 
     wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  have any of the values defined in the specification, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions comprising compounds of formula I, intermediates and processes useful for preparing compounds of formula I, and methods comprising inhibiting tumor growth or treating cancer by administering one or more compounds of formula I.

BACKGROUND OF THE INVENTION

[0001] A listing of human cancers for which chemotherapy has exerted apredominant role in increasing life span, approaching normal lifeexpectancy, includes Burkiti's lymphoma, acute lymphocytic leukemia andHodgkin's disease, along with about 10-15 other tumor types. Forexample, see A. Golden et al., Eur. J. Cancer. 11, 129 (1981) (Table 1).While the cure rate of these cancers illustrates the level of success ofscreening systems in selecting antitumor agents that are effective inman, these responsive tumors represent only a small fraction of thevarious types of cancer and, notably, there are relatively few drugshighly active against solid tumors such as ovarian cancer, breastcancer, lung cancer and the like. Such drugs include cyclophosphamide,adriamycin, 5-FU, hexamethylmelamine and the like. Thus, patients withmany types of malignancies remain at significant risk for relapse andmortality.

[0002] After relapse, some patients can be reinduced into remission withtheir initial treatment regimen. However, higher doses of the initialchemotherapeutic agent or the use of additional agents are frequentlyrequired, indicating the development of at least partial drugresistance. Recent evidence indicates drug resistance can developsimultaneously to several agents, including ones to which the patientwas not exposed. The development of multipledrug resistant (mdr) tumorsmay be a function of tumor mass and constitutes a major cause oftreatment failure. To overcome this drug resistance, high-dosechemotherapy with or without radiation and allogenic or autologous bonemarrow transplantation can be employed. The high-dose chemotherapy mayemploy the original drug(s) or be altered to include additional agents.The development of new drugs, non-cross resistant with mdr phenotypes,is required to further the curative potential of current regimens and tofacilitate curative interventions in previously treated patients.

[0003] The in vitro anti-tumor activity of a novel class of naturalproducts called illudins has been examined by Kelner, M. et al., CancerRes. A, 3186 (1987). Illudin M was purified and submitted for evaluationto the National Cancer Institute Division of Cancer Treatment (NCI DCT)in vivo drug screening program. Illudin M significantly increased thelife span of rats with Dunning leukemia, but had a low therapeutic indexin solid tumor systems. The extreme toxicity of illudins has preventedany applications in human tumor therapy. Recently, synthetic analogs ofthe illudins have been developed which exhibit promising antitumoractivity, including those analogs disclosed in U.S. Pat. Nos. 5,439,936and 5,523490.

[0004] However, despite these developments, there exists a continuingneed for chemotherapeutic agents which inhibit tumor growth, especiallysolid tumor growth, and which have an adequate therapeutic index to beeffective for in vivo treatment.

SUMMARY OF THE INVENTION

[0005] The invention provides a compound of formula I:

[0006] wherein

[0007] R₁ is hydrogen, hydroxy, mercapto, amino, halo, carboxy, nitro,or —(CH₂)_(n)—(X)—(Y);

[0008] n is 0 to 4;

[0009] X is oxy (—O—), thio (—S—), —N(R_(a))—, or absent;

[0010] Y is (C₃-C₆)cycloalkyl, aryl, heteroaryl, a saccharide, an aminoacid, a peptide, or a 1 to 15 membered branched or unbranched carbonchain optionally comprising 1, 2, or 3 non-peroxide oxy, thio, or—N(R_(a))—; wherein said chain may optionally be substituted on carbonwitt 1, 2, or 3, oxo (═O), hydroxy, carboxy, halo, mercapto, nitro,—N(R_(b))(R_(c)), (C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides,amino acids, or peptides; and wherein said chain may optionally besaturated or unsaturated;

[0011] R₂ is carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)N_(d)R_(e), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl substituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(d)R_(e);

[0012] R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio,aryl, heteroaryl, aryloxy, or heteroaryloxy;

[0013] R₄ is hydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy,or (C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy;

[0014] R₆ is hydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(f)R_(g);

[0015] R_(a) is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl orbenzyl; and

[0016] R_(b), R_(d), R_(e), R_(f) and R_(g) are each independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) andR_(c), R_(d) and R_(e), or R_(f) and R_(g), together with the nitrogento which they are attached, are pyrrolidino, piperidino, or morpholino;

[0017] wherein any aryl, heteroaryl, aryloxy, or heteroaryloxy of Y, orR₃ may optionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy,halo, carboxy, mercapto, nitro, or —N(R_(h))(R_(j)); wherein each R_(h)and R_(j) is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; or R_(h) and R_(j) together with the nitrogen to whichthey are attached are pyrrolidino, piperidino, or morpholino;

[0018] or a pharmaceutically acceptable salt thereof.

[0019] The invention also provides a compound of formula I wherein: R₁is —(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio, —N(R_(a))—, orabsent; Y is a monoprotected amino acid, a diprotected amino acid, apeptide, or a 1 to 15 membered branched or unbranched carbon chainoptionally comprising 1, 2, or 3 non-peroxide oxy, thio, or —N(R_(a))—;wherein said chain is substituted with 1, 2, or 3 peptides; and whereinsaid chain may optionally be saturated or unsaturated; R₂ is hydrogen 6r(C₁-C₆)alkyl; R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy; R₆ ishydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; and R_(b), R_(c), R_(d), R_(e), R_(f)and R_(g) are each independently hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) and R_(c), R_(d) and R_(e),or R_(f) and R_(g), together with the nitrogen to which they areattached, are pyrrolidino, piperidino, or morpholino; wherein any aryl,heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ may optionally besubstituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl,halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto, nitro, or—N(R_(h))R_(j)); wherein each R_(h) and R_(j) is independently hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(h) and R_(j)together with the nitrogen to which they are attached are pyrrolidino,piperidino, or morpholino; or a pharmaceutically acceptable salt thereofPreferably, Y is (C₁-C₆)alkyl substituted with a peptide.

[0020] The invention also provides a compound of formula I wherein: R₁is hydrogen, hydroxy, mercapto, amino, halo, carboxy, nitro, or—(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio, —N(R_(a))—, or absent;Y is (C₃-C₆)cycloalkyl, aryl, heteroaryl, a saccharide, an amino acid, apeptide, or a 1 to 15 membered branched or unbranched carbon chainoptionally comprising 1, 2, or 3 non-peroxide oxy, thio, or —N(R_(a))—;wherein said chain may optionally be substituted on carbon with 1, 2, or3, oxo, hydroxy, carboxy, halo, mercapto, nitro, —N(R_(b))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; and wherein said chain may optionally be saturated orunsaturated; R₂ is hydrogen or (C₁-C₆)alkyl; R₃ is hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl, heteoaryl, aryloxy,or heteroaryloxy; R₄ is hydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy,(C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together areethylenedioxy; R₆ is carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,—C(═O)NR_(f)R_(g), a saccharide, an amino acid, a peptide, or(C₁-C₆)alkyl substituted by 1 or 2 (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy,carboxy, amino acids, peptides, saccharides, or —C(═O)NR_(f)R_(g); R_(a)is hydrogen, (C₃-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; and R_(b),R_(c), R_(d), R_(e), R_(f) and R_(g) are each independently hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) and R_(c),R_(d) and R_(e), or R_(f) and R_(g), together with the nitrogen to whichthey are attached, are pyrrolidino, piperidino, or morpholino; whereinany aryl heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ mayoptionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁ ₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl,hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto,nitro, or —N(R_(h))(R_(j)); wherein each R_(h) and R_(j) isindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl;or R_(h) and R_(j) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, or morpholino; or a pharmaceuticallyacceptable salt thereof.

[0021] The invention also provides dimeric compounds comprising twocompounds of formula (I), connected by a linker. The linker can be, forexample, an alkyl or ester based linker group. Examples of suitablelinkers include —(CH₂)_(p)—O— (CH₂)_(q)—, CH₂)_(r)—, and—CH₂—S—CH₂C(O)—(CH₂)₂—OC(O)CH₂—SCH₂—; wherein p and q are eachindividually an integer from 1 to 8, inclusive; and r is an integer from1 to 16, inclusive. Preferably, r is an integer from 1 to 8, inclusive.As would be apparent to one skilled in the art, other linkers ofapproximately the same length can also be used. Two compounds of formulaI can conveniently be linked, for example, by replacing R₁, R₃, R₄ orR₅, independently, on each compound of formula I, with the bifunctionallinker. When the linkage is through R₁, the linker is preferably—CH₂—O—CH₂— or —CH₂—SCH₂C(O)—OCH₂)₂—O—C(O)CH₂—S—CH₂—.

[0022] Compounds of the invention are useful as antineoplastic agents,i.e., to inhibit tumor cell growth in vitro or in vivo, in mammalianhosts, such as humans or domestic animals, and are particularlyeffective against solid tumors and multi-drug resistant tumors. Thus,the invention provides a method comprising inhibiting cancer cells, bycontacting said cells, in vitro or in vivo, with an effective amount ofa compound of formula I. The invention also provides a therapeuticmethod comprising treating cancer (i.e., inhibiting tumor cell growth)by administering a compound of formula I to a mammal (e.g. a human) inneed of such therapy.

[0023] The present compounds may be targeted to a particular tumor byattacking the compound to a reagent which is capable of binding to atumor-associated antigen. The antigen may be located on a tumor or inthe tumor cell area. Suitable reagents include polyclonal and monoclonalantibodies. The compound-reagent complex or conjugate may furthercomprise a linker (e.g. linker as described hereinabove) for attachingthe compound to the reagent. Accordingly, the invention also provides acompound comprising a compound of formula I and a reagent (e.g. apolyclonal or monoclonal antibody) which is capable of binding to atumor-associated antigen.

[0024] The present invention also provides a pharmaceutical composition(e.g. a pharmaceutical unit dosage form), comprising one or morecompounds of the invention in combination with a pharmaceuticallyacceptable diluent or carrier.

[0025] The invention also provides a compound of the invention (e.g. acompound of formula I, a dimer thereof, or a conjugate comprising acompound of formula I and a reagent that is capable of binding to atumor-associated antigen) for use in medical therapy (preferably for usein treating cancer, e.g. solid tumors), as well as the use of a compoundof the invention for the manufacture of a medicament useful for thetreatment of cancer, e.g. solid tumors.

[0026] The invention also provides processes and novel intermediatesdisclosed herein that are useful for preparing compounds of theinvention. Some of the compounds of the invention are useful to prepareother compounds of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 shows representative compounds of the invention (compounds1 to 9) and intermediate compounds 10 or 11.

[0028]FIG. 2 shows representative compounds of the invention (compounds13 to 16).

DETAILED DESCRIPTION

[0029] The following definitions are used, unless otherwise described:halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, etc.denote both straight and branched groups; but reference to an individualradical such as “propyl” embraces only the straight chain radical, abranched chain isomer such as “isopropyl” being specifically referredto. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclicradical having about nine to ten ring atoms in which at least one ringis aromatic. Heteroaryl encompasses a radical attached via a ring carbonof a monocyclic aromatic ring containing five or six ring atomsconsisting of carbon and one to four non-peroxide oxygen, sulfur, orN(R_(y)) wherein R_(y) is absent or is H, O, (C₁-C₄)alkyl, phenyl orbenzyl, as well as a radical of an ortho-fused bicyclic heterocycle ofabout eight to ten ring atoms derived therefrom, particularly abenz-derivative or one derived by fusing a propylene, trimethylene, ortetramethylene diradical thereto.

[0030] The term “inhibit” or “inhibiting” means decreasing tumor cellgrowth rate from the rate which would occur without treatment, and/orcausing tumor mass to decrease. Inhibiting also includes causing acomplete regression of the tumor. Thus, the present analogs tan eitherbe cytostatic or cytotoxic to tumor cells.

[0031] The method of the invention can be practiced on any mammal havinga susceptible cancer, i.e., a malignant cell population or tumor.Compounds of the invention are effective on human tumors in vivo as wellas on human tumor cell lines in vitro. The present compounds may beparticularly useful for the treatment of solid tumors for whichrelatively few treatments are available. Such tumors include epidermoidand myeloid tumors, acute (AML) or chronic (CML). Such tumors alsoinclude, nonsmall cell, squamous, liver, cervical, renal, adrenal,stomach, esophageal, oral and mucosal tumors, as well as lung, ovarian,breast and colon carcinoma, and melanomas (including amelanoticsubtypes). The present compounds can also be used against endometrialtumors, bladder cancer, pancreatic cancer, lymphoma, Hodgkin's disease,prostate cancer, sarcomas and testicular cancer as well as againsttumors of the central nervous system, such as brain tumors,neuroblastomas and hematopoietic cell cancers such as B-cellleukemia/lymphomas, myelomas, T-cell leukemia/lymphomas, small cellleukemia/lymphomas, as well as null cell, sezary, monocytic,myelomonocytic and Hairy cell leukemia. These leukemia/lymphomas can beeither acute (ALL) or chronic (CLL).

[0032] The term “saccharide” includes monosaccharides, disaccharides,trisaccharides and polysaccharides. The term includes glucose, sucrosefructose and ribose, as well as deoxy sugars such as deoxyribose and thelike. Saccharide derivatives can conveniently be prepared as describedin International Patent Applications Publication Numbers WO 96/34005 and97/03995. A saccharide can conveniently be linked to the remainder of acompound of formula I through an ether bond.

[0033] The term “amino acid,” comprises the residues of the naturalamino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or Lform, as well as unnatural amino acids (e.g. phosphoserine,phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, citruline, α-methyl-alanine,para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine,and tert-butylglycine). The term also comprises natural and unnaturalamino acids bearing a conventional amino protecting group (e.g. acetylor benzyloxycarbonyl), as well as natural and unnatural amino acidsprotected at the carboxy terminus (e.g. as a (C₁-C₆)alkyl, phenyl orbenzyl ester or amide; or as an a-methylbenzyl amide). Other suitableamino and carboxy protecting groups are known to those skilled in theart (See for example, T. W. Greene, Protecting Groups In OrganicSynthesis; Wiley: New York, 1981, and references cited therein). Anamino acid can be linked to the remainder of a compound of formula Ithrough the carboxy terminus, the amino terminus, or through any otherconvenient point of attachment, such as, for example, through the sulfurof cysteine.

[0034] The term “peptide” describes a sequence of 2 to 25 amino acids(e.g. as defined hereinabove) or peptidyl residues. The sequence may belinear or cyclic. For example, a cyclic peptide can be prepared or mayresult from the formation of disulfide bridges between two cysteineresidues in a sequence. A peptide can be linked to the remainder of acompound of formula I through the carboxy terminus, the anmno terminus,or through any other convenient point of attachment, such as, forexample, through the sulfir of a cysteine. Preferably a peptidecomprises 3 to 25, or 5 to 21 amino acids. Peptide derivatives can beprepared as disclosed in U.S. Pat. Nos. 4,612,302; 4,853,371; and4,684,620, or as described in the Examples hereinbelow. Peptidesequences specifically recited herein are written with the aminoterminus on the left and the carboxy terminus on the right.

[0035] It has been shown that certain peptides specifically bind tospecific tumor-associated antigens in a manner analogous to the bindingof antibodies to such antigens. See Arap et al. Science, 1998, 279,5349, 377-380. Thus, pharmaceutical agents which comprise a peptide thatcapable of binding to specific receptors on tumor cells, can bedelivered preferentially to such tumor cells. As a result, one preferredembodiment of the invention provides a compound of formula I comprisinga peptide capable of specifically binding to a tunor-associated antigen.

[0036] Preferred peptides include -Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys(SEQ ID NO:1), -Cys-Asp-Gly-Arg-Cys (SEQ ID NO:2), and-Cys-Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ IDNO:3).

[0037] It will be appreciated by those skilled in the art that compoundsof the invention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine anti-tumor activity using Test Aor Test B, described hereinbelow, or using other tests which are wellknown in the art.

[0038] In preferred compounds of formula I, the absolute stereochemistryat the carbon bearing R₄ and R₅ is (R).

[0039] Specific values listed below for radicals, substituents, andranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents Specifically, (C₁-C₄)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, or sec-butyl; (C₁-C₆)alkyl can be methyl,ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl,or hexyl; (C₁-C₈)alkyl can be methyl, ethyl, propyl, isopropyl, butyl,iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, septyl, or octyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₁-C₄)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, or sec-butoxy; (C₁-C₆)alkoxy can be methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,or hexyloxy; (C₂-C₆)alkenyl can be vinyl, alkyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 3-methyl-2-butenyl,3-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, or 4-methyl-3-pentenyl; (C₁-C₆)alkanoyl can be acetyl,propanoyl or butanoyl; halo(C₁-C₆)alkyl can be iodomethyl, bromomethyl,chloromethyl, fluoromethyl, trichloromethyl, trifluoromethyl,2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl;hydroxy(C₁-C₆)alkyl can be hydroxymethyl, 1-hydroxyethyl,2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl,1-hydroxyhexyl, or 6-hydroxyhexyl; (C₁-C₆)alkoxycarbonyl can bemethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C₁-C₆)alkylthiocan be methylthio, ethylthio, propylthio, isopropylthio, butylthio,isobutytltijo, pentylthio, or hexylthio; (C₁-C₆)alkanoyloxy can beformyloxy, acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy,pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl;and heteroaryl can be fliryl, imidazolyl, triazolyl, triaiinyl, oxazoyl,isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or itsN-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or itsN-oxide).

[0040] A specific value for R₁ is hydroxy, halo, carboxy, aryl,heteroaryl, a saccharide, an amino acid, or a peptide.

[0041] Another specific value for R₁ is CH₂)_(n)—(X)—(Y); wherein n is 0to 4; X is oxy, thio, —N(R_(a))—, or absent; and Y is a peptide, or(C₁-C₆)alkyl substituted with a peptide.

[0042] Another specific value for R₁ is hydrogen or (C₁-C₆)alkyl,optionally substituted with 1 or 2 hydroxy, halo, methoxy or ethoxy.

[0043] Another specific value for R₁ is —(CH₂)_(n)C(X)—(Y); X is oxy,thio, or —N(R,)—; and Y is CH₂OC(O)(C₁-C₄)alkyl,CH₂C(O)—O—(CH₂)₂—O—C(O)CH₂SH, (CH₂)₂—O—(CH₂)₂halo,(C₁-C₄)alkyl-O(C₁-C₄)alkyl, CH₂CO₂(C₁-C₄)alkyl, CH₂CO₂H,aryl(C₁-C₄)akyl, a saccharide, an amino acid, or (CI-Cg)alkyl optionallysubstituted with 1 or 2 hydroxy or halo; wherein any aryl or heteroarylof Y may optionally be substituted with 1 or 2 hydroxy, halo,(C₁-C₄)alkyl or (C₁-C₄)alkolxy.

[0044] Another specific value for R₁ is —CH₂—(X)—(Y), wherein X is oxy,thio, or —N(R_(a))—; and Y is (C₁-C₈)alkyl optionally substituted with 1or 2 hydroxy, halo, carboxy, oxo, mercapto, —N(R_(b))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; wherein any aryl or heteroaryl of Y may optionally besubstituted by 1, 2, or 3(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl,halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto, nitro, or—N(R_(h))(R_(j)); wherein each R_(h) and R_(j) is independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(h) andR_(j) together with the nitrogen to which they are attached form apyrrolidino, piperidino, or morpholino radical.

[0045] Another specific value for R₁ is 4CH₂)_(n)—(X)—(Y); n is 1 or 2;X is oxy, thio, or —N(R_(a))—; and Y is (C₁-C₆)alkyl or (C2-C₆)alkenyl,optionally substituted with 1 or 2 oxo, hydroxy, carboxy, halo,mercapto, nitro, -N(R ,)(R), (C₃-C₆)cycloalkyl, aryl, heteroaryl,saccharides, amino acids, or peptides; wherein any aryl or heteroaryl ofY may optionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy,halo, carboxy, mercapto, nitro, and —N(R_(h))(R_(j)); wherein each R_(h)and R_(j) is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl and benzyl; or R_(h) and R_(j) together with the nitrogen towhich they are attached form a pyrrolidino, piperidino, or morpholinoradical.

[0046] Another specific value for R₁ is —CH₂-[sulfur-linked-cysteine]-R_(x) wherein R_(x) is an amino acid or a peptide comprising2 to 24 amino acids.

[0047] Another specific value for R₁ is—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) wherein R_(x) is an amino acidor a peptide comprising 2 to 24 amino acids.

[0048] Another specific value for R₁ is—CH₂-[sulfur-linked-glutathione].

[0049] Another specific value for R₁ is2-[(R)-α-methylbenzyl-aminocarbonyl]-2-(acylamino)ethylthiomethyl.Preferably, the 2-position of the ethyl group has the (S) configurationof cysteine.

[0050] A more specific value for R₁ is hydrogen, methyl, hydroxymethyl,methoxymethyl, or acetoxymethyl.

[0051] A specific value for R₂ is hydroxymethyl, methoxymethyl, oracetoxymethyl.

[0052] Another specific value for R₂ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, or —C(═O)NR_(d)R_(e).

[0053] Another specific value for R₂ is (C₁-C₆)alkyl substituted by 1 or2 hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids,peptides, saccharides, or —C(═O)NR_(d)R_(e).

[0054] Another specific value for R₂ is —CH₂-[sulfur-linked-cysteine]-R_(x) wherein R_(x) is an amino acid or a peptide comprising2 to 24 amino acids.

[0055] Another specific value for R₂ is—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) wherein R_(x) is an amino acidor a peptide comprising 2 to 24 amino acids.

[0056] Another specific value for R₂ is —CH₂-[sulfur-linked-glutathione].

[0057] A specific value for R₃ is hydrogen.

[0058] A specific value for R₄ is methyl.

[0059] A specific value for R₅ is hydroxy.

[0060] A specific value for R₆ is hydrogen.

[0061] A specific value for R₆ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, or —C(═O)NR_(f)R_(g).

[0062] A specific value for R₆ is (C₁-C₆)alkyl optionally substituted by1 or 2 hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkinoyloxy, carboxy, amino acids,peptides, saccharides, or —C(═O)NR_(f)R_(g).

[0063] Another specific value for R₆ is —CH₂-[sulfur-linked-cysteine]-R_(x) wherein R_(x) is an amino acid or a peptide comprising2 to 24 amino acids.

[0064] Another specific value for 6 is—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) wherein R_(x) is an amino acidor a peptide comprising 2 to 24 amino acids.

[0065] A more specific value for R_(x) is methyl or hydroxymethyl.

[0066] Specifically, R_(x) can be a peptide comprising 4 to 20 aminoacids.

[0067] Another specific value for R_(x) is -Leu-Gly-Phe, -Phe-Leu-Gly,-Leu-Leu-Phe, -Gly-Phe, or -Leu.

[0068] Another specific value for R_(x) is-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO:4), -Asp-Gly-Arg-Cys (SEQ IDNO:5), or -Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQID NO:5).

[0069] A specific compound of the invention is a compound of formula Iwherein R₂ is (C₁-C₆)alkyl, substituted with hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, or carboxy; R₃ is H, (C₁C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; R₅ is hydroxy; and R₆ is (C₁-C₆)alkyl,optionally substituted with hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy,or carboxy; or a pharmaceutically acceptable salt thereof.

[0070] Another specific compound of the invention is a compound offormula I wherein R₁ is hydrogen or (C₁-C₆)alkyl, optionally substitutedwith hydroxy, halo, methoxy, ethoxy, or acetoxy; R₂ is hydroxymethyl,methoxymethyl, or acetoxymethyl; R₃ is hydrogen; R₄ is methyl; R₅ ishydroxy; and R₆ is methyl or hydroxymethyl; or a pharmaceuticallyacceptable salt thereof.

[0071] Another specific compound of the invention is a compound offormula I wherein R₁ is —(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio,—N(R_(a))—, or absent; Y is a monoprotected amino acid or a diprotectedamino acid; and R₂ is hydrogen or (C₁-C₆)alkyl; or a pharmaceuticallyacceptable salt thereof.

[0072] Another specific compound of the invention is a compound offormula I wherein R₁ is —CH₂-[sulfur-linked -N-acylcysteine],(S)-2-[(R)-α-methylbenzylaiinocarbonyl]-2-(acylamino)ethylthiomethyl, or(R)-2-[(R)-α-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl;and R₂ is hydrogen or (C₁-C₆)alkyl; or a pharmaceutically acceptablesalt thereof.

[0073] A preferred compound is a compound of formula I wherein R₄ ishydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy or acetoxy; wherein theabsolute stereochemistry at the carbon bearing R₄ and R₅ is (R); or apharmaceutically acceptable salt thereof.

[0074] Processes for preparing compounds of the invention are providedas further embodiments of the invention and are illustrated by thefollowing procedures in which the meanings of the generic radicals areas given above unless otherwise qualified.

[0075] The compounds of the present invention may be derived fromilludin S, hydroxymethyl acylfilvene (HAF, i.e., a compound of formula(1) wherein R₁ is CH₂OH, R₂ is CH₃, R₃ is hydrogen, R₄ is CH₃, and R₅ isCH₃) and fulvene (i.e., a compound of formula (1) wherein R₁ is H, R₂ isCH₃, R₃ is H, R₄ is CH₃, and R₅ is CH₃) the syntheses of which are knownin the art (see e.g., WO 91/04754; WO 94/18151).

[0076] A compound of formula I wherein R₂ is hydroxymethyl can beprepared by oxidation of a corresponding compound of formula I whereinR₂ is methyl. The oxidation can conveniently be carried out usingselenium dioxide and tert-butyl hydroperoxide under conditions similarto those described in Example 1.

[0077] A compound of formula I wherein R₁ is hydroxymethyl can beprepared from a corresponding compound of formula I wherein R₁ ishydrogen by treatment with paraformaldehyde and sulfuric acid. Thereaction can conveniently be carried out under conditions similar tothose described in Example 2.

[0078] A compound of formula I wherein R₂ is acetoxymethyl can beprepared by acylation of a corresponding compound of formula I whereinR₂ is hydroxymethyl. The acylation can conveniently be carried out usingacetic anhydride, under conditions similar to those described in Example3.

[0079] A compound of formula I wherein R₂ is methoxymethyl can beprepared by reacting a corresponding compound of formula I wherein R₂ ishydroxymethyl with methyl iodide and silver oxide. The reaction canconveniently be carried out under conditions similar to those describedin Example 7.

[0080] A compound of formula I wherein R₁ is methoxymethyl can beprepared by reacting a corresponding compound of formula I wherein R₁ ishydroxymethyl with methanol and sulfuric acid. The reaction canconveniently be carried out under conditions similar to those describedin Example 8, sub-part a.

[0081] A compound of formula I wherein R₁ is—CH₂-[sulfur-linked-cysteine] can be prepared by reacting acorresponding compound of formula I wherein R₁ is hydroxymethyl bycoupling with cysteine. The reaction can conveniently be carried outunder conditions similar to those described in Example 10.

[0082] A compound of formula I wherein R₁ is—CH₂-[sulfur-linked-N-acylcysteine] can be prepared by reacting acorresponding compound of formula I wherein R₁ is hydroxymethyl bycoupling with N-acylcysteine. The reaction can conveniently be carriedout under conditions similar to those described in Example 11.

[0083] A compound of formula I wherein R₁ is2-[(R)-α-methylbenzyl-aminocarbonyl]-2acylamino)ethylthiomethyl can beprepared by reacting a corresponding compound of formula I wherein R₁ is—CH₂-[sulfur-linked-N-acylcysteine] with α-methylbenzylamine. Thereaction can conveniently be carried out under conditions similar tothose described in Example 12.

[0084] A compound of formula I wherein R₁ is—CH₂-[sulfur-linked-glutathione] can be prepared by reacting acorresponding compound of formula I wherein R₁ is hydroxymethyl withglutathione. The reaction can conveniently be carried out underconditions similar to those described in Example 14.

[0085] A compound of formula I wherein R₁ is—CH₂-[sulfur-linked-cysteine]-R_(x), or—CH₂-[sulfur-linked-N-acylcysteine]-R_(x) can be prepared by reacting acorresponding compound of formula I wherein R₁ is—CH₂-[sulfur-linked-cysteine] or —CH₂-[sulfur-linked-N-acylcysteine]with the requisite amino acid or peptide (R). The reaction canconveniently be carried out under conditions similar to those describedin Example 15.

[0086] Compounds of the invention can also be prepared using techniquesand intermediates similar to those described by T. McMorris et al.Tetrahedron, 1997, 53, 44, 14579-14590; T. McMorris et al. J. Org. Chem.1997, 62, 3015-3018; T. McMorris et al. Chem. Comm. 1997, 315-316; andT. McMorris et al. Experentia 1996, 52, 75-80; and those disclosed inU.S. Pat. No. 5,439,942; U.S. Pat. No. 5,439,936; U.S. Pat. No.5,523,490; U.S. Pat. No. 5,536,176; WO 91/04754; WO 94/18151 and WO98103458.

[0087] In cases where compounds are sufficiently basic or acidic to formstable nontoxic acid or base salts, administration of the compounds assalts may be appropriate. Examples of pharmaceutically acceptable saltsare organic acid addition salts formed with acids which form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

[0088] Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

[0089] The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of dosage forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

[0090] Thus, the present compounds may be systemically administered,e.g., orally, in combination with a pharmaceutically acceptable vehiclesuch as an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

[0091] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive compound may be incorporated into sustained-release preparationsand devices.

[0092] The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

[0093] The pharmaceutical dosage forms suitable for injection orinfusion can include sterile aqueous solutions or dispersions or sterilepowders comprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infisible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersionsorby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0094] Sterile injectable solutions are prepared by incorporating theactive compound in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desired aingredient present in the previously sterile-filtered solutions.

[0095] For topical administration, the present compounds may be appliedin pure form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

[0096] Useful solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

[0097] Thickeners such as synthetic polymers, fatty acids, fatty acidsalts and esters, fatty alcohols, modified celluloses or modifiedmineral materials can also be employed with liquid carriers to formspreadable pastes, gels, ointments, soaps, and the like, for applicationdirectly to the skin of the user.

[0098] Examples of useful dermatological compositions which can be usedto deliver the compounds of formula I to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

[0099] Useful dosages of the compounds of Figure (I) can be determinedby correlating their in vitro activity, and in vivo activity in animalmodels, such as murine or dog models as taught for illudin analogs suchas those of U.S. Pat. Nos. 5,439,936 and 5,523,490, to activity inhigher mammals, such as children and adult humans as taught, e.g., inBorch et al. (U.S. Pat. No. 4,938,949).

[0100] The therapeutically effective amount of analog necessarily varieswith the subject and the tumor to be treated. It has been found thatrelatively high doses of representative compounds of formula I can beadministered due to the decreased toxicity compared to illudin S and M.For example, the maximum tolerated dose of Illudin S is about 250 ug/kg,whereas compound 2 can be chronically administered at 35 mg/kg withouttoxicity. A therapeutic amount between 30 to 112,000 μg per kg of bodyweight is especially effective for intravenous administration while 300to 112,000 μg per kg of body weight is effective if administeredintraperitoneally. As one skilled in the art would recognize, the amountcan be varied depending on the method of administration. The amount ofthe compound, or an active salt or derivative thereof, required for usein treatment will vary not only with the particular salt selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician.

[0101] The compound can conveniently be administered in unit dosageform; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg,most conveniently, 50 to 500 mg of active ingredient per unit dosageform.

[0102] Ideally, the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 0.5 toabout 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 toabout 30 μM. This may be achieved, for example, by the intravenousinjection of a 0.05 to 5% solution of the active ingredient, optionallyin saline, or orally administered as a bolus containing about 1-100 mgof the active ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

[0103] The desired dose may conveniently be presented in a single doseor as divided doses administered at appropriate intervals, for example,as two, three, four or more sub-doses per day.

[0104] The cytotoxic and anti-tumor properties of a compound of theinvention can be determined using pharmacological models which are wellknown to the art, or using Test A and Test B described below.

[0105] Test A. In Vitro Studies

[0106] To assess cytotoxic effects, various concentrations of compoundsof the invention were added to cultures of MV522 (human lung carcinomacell line), HL60 (myeloid leukemia cells), and 8392 (B-cellleukemia/lymphoma) cells for 48 hours, then cell grow/viability wasdetermined by trypan blue exclusion. As an alternative to 48 hourcontinuous exposure studies, cells were plated in liquid culture in 96well plates, exposed to various concentrations of compounds of theinvention for 2 hours, pulsed with [³H]-thymidine for one to two hoursand harvested onto filter papers. The filter papers were added to vialscontaining scintillation fluid and residual radioactivity determined ina beta (scintillation) counter.

[0107] Data from Test A, for representative compounds of the invention,is shown in Table 1. Values are reported as mean +I standard deviation;units are nanomoles per liter, and NT signifies not tested. TABLE 1 2hour IC₅₀ (nm/l) Compound MV522 HL60 8392 1 2,640 ± 360  NT 37,000 ±2,300 2 11,300 ± 1,500 NT NT 3 19,600 ± 9,700 15,600 ± 4,600  62,000 ±3,600 4 20,400 ± 6300  >40,000 NT 5 24,000 ± 6,100 38,400 ± 19,000 NT 6NT NT NT 7  7,700 ± 3,500 6,000 ± 1,200 NT 8 >80,000 >80,000 NT9 >50,000 49,800 ± 20,000 NT 13  10,000 ± 1,800 NT NT 14  3,050 ± 550 NT NT 15  NT NT NT

[0108] Test B. In Vivo Studies

[0109] Several representative compounds of the invention were chosen forin vivo studies, The anticancer agent mitomycin C was used as apharmaceutical control. Drug therapy was started 10 days afterinoculation on a daily basis via IP route for 5 consecutive days. Theanimals were monitored for 3 weeks after start of therapy. The MTD wasreached for the control agent mitomycin C but not for compounds 1 or 2.

[0110] BALB/c nu/nu 4-week old female mice weighing 18-22 g wereobtained from Simonsen, Inc. (Gilroy, Calif.) and maintained in theathymic mouse colony of the University of California (San Diego, Calif.)under pathogen free conditions using HEPA filter hoods. Animals wereprovided with sterilized food and water ad libitum in groups of 5 inplastic cages vented with polyester fiber filter covers. Clean,sterilized gowns, glove, face masks, and shoe and hood covers were wornby all personnel handling the animals. All studies were conducted inaccordance with guidelines of the NIH “Guide for Care and Use ofAnimals” and approved by the University Institutional Animal Care andUse Committee (Protocol 3-006-2)

[0111] The MV522 lung carcinoma line used for xenograft studies wasderived as described by Kelner et al. (Anticancer Res., 15: 867-872;873-878 (1995)) and maintained in antibiotic-free RPMI 1640 (Mediatech,Hemdon, Va.) supplemented with 10% fetal bovine serum and 2 mM glutaminein 37° C. humidified carbon dioxide incubator.

[0112] Mice were randomized into treatment groups of five animals eachfor initial studies and groups of 16-20 animals for confirming analogueefficacy. Each animal was earmarked and followed individually throughoutthe experiments. Mice received s.c. injections of the parental cell lineMV522 using 10 million cells/inoculation over the shoulder. Ten daysafter s.c. implantation of the MV522 cells, when s.c. tumors wereapproximately 3×3 mm in size, animals received the desired drug anddosage. The effect of the compound on life span was calculated frommedian survival.

[0113] Although MV522 cells kill mice by metastases, primary s.c. tumorgrowth over the shoulder was monitored starting on the first day oftreatment and at weekly intervals thereafter. Tumor size was measured intwo perpendicular diameters. Tumor weights were estimated according tothe formula w=(width)²×length/2). Relative weights (R F) were calculatedto standardized variability in tumor size among test groups atinitiation of treatment using the formula RW Wt/wi, where Wi is thetumor weight for a given animal at beginning of drug treatment and Wt istumor weight at a subsequent time. Animals were necropsied, and organswere examined for evidence of metastases.

[0114] Comparison of survival curves between groups of animals was bythe method of Kaplan and Meier. For comparison of relative tumor weightsbetween multiple groups of animals, ordinary ANOVA followed byTukey-Kramer multiple Comparison post ANOVA analysis was performed(Kelner et al. (Anticancer Res., 15: 867-872; 873-878 (1995)).Probability values (p) less than 0.05 were considered statisticallysignificant.

[0115] Data from Test B, for representative compounds of the invention,is shown in Table 2. Data was collected using eight animals in thecontrol studies and four animals in each compound study. Valuesrepresent relative tumor weight or Wt/Wi (day ten by definition is 1.0).Drug was administered i.p. on days 10, 11, 12, 13, and 14 (QD×5 days).TABLE 2 Compound Day 17 Day 24 Day 31 control (saline) 4.3 ± 0.7 7.0 ±1.3 11.8 ± 3.0   1 (14 mg/kg) 1.4 ± 0.7 7.0 ± 1.3 11.8 ± 3.0   2 (35mg/kg) 2.1 ± 0.2 3.3 ± 0.3 4.0 ± 0.9 13 (32 mg/kg) 2.3 ± 0.5 4.1 ± 0.84.7 ± 1.5 14 (28 mg/kg) 3.2 ± 0.6 4.1 ± 0.8 6.0 ± 1.8

[0116] The data in Tables 1 and 2 shows that representative compounds ofthe invention are potent cytotoxic and anti-tumor agents.

[0117] The invention will now be illustrated by the followingnon-limiting Examples, wherein, unless other wise stated: Melting pointsare uncorrected; ¹H and ¹³C NMR spectra were measured at 300 and 75 MHz;High resolution mass spectra were determined at the University ofMinnesota Mass Spectrometry Service Laboratory; Chromatography wasperformed of silica gel (Davisil 230425 mesh, Fisher Scientific), withethyl acetate:hexanes as the eluent; Analytical TLC was carried out onWhatman 4420 222 silica gel plates; and Reactions were routinelymonitored by TLC. Synthesis of illudin S, hydroxymethylacylfulvene(HMAF) and fulvene were prepared as previously described (see, e.g., WO91/04754; WO 94/18151).

EXAMPLES Example 1 Compound I (Formula I Wherein R₁ is Hydrogen; R₂ isHydroxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0118] To a stirred solution of acylfulvene (6.9 g, 32 mmol) in 7.0 mlof EtOAc, were added 99% Selenium dioxide (1.75 g, 15.8 mmol), and 90%tert-butyl hydroperoxide (6 ml, d 0.901, 60 mmol). The mixture wasstirred at room temperature for 24 hr and then partitioned between EtOAcand saturated sodium sulfite (3×3 ml), followed by saturated brine. Theorganic extract was then dried over MgSO₄. After concentration, thecrude product was chromatographed to give 922.5 mg of the title compound(12%) as a yellow-orange gum, and 5.8 g of Acylfulvene (84%); ¹H NMR(CDCl₃) δ 0.76 (ddd, 1H), 1.14 (ddd, 1H), 1.27 (ddd, 1H), 1.36 (s, 3H),1.54 (ddd, 1H), 2.03 (s, 3H), 4.02 (s, 1H), 4.56 (s, 2H), 6.67 (s, 1H),7.29 (s, 1H); ¹³C NMR (CDCl₃) δ 197.9, 161.5, 146.0, 140.2, 132.9,127.0, 119.8, 76.6, 59.9, 37.5, 27.7, 17.0, 14.7,9.9; MS m/e 232 (M⁺);UV λmax (EtOH) 327.2 nm (ε 7631).

Example 2 Compound 2 (Formula I Wherein R₁ is Hydroxymethyl; R₂ isHydroxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0119] Paraformaldehyde (36.3 g) was added to a solution of dilute H₂SO₄(1.5 M, 275 ml), and MECO (300 ml). The mixture was stirred and heatedto dissolve all the solid. To the cooled (0° C.) solution compound 1(922.50 mg, 3.98 mmol) was added, the resulting solution was stirred andallowed to warm to room temperature. After 24 hr, the orange-yellowmixture was extracted with EtOAc (2×300 ml) and the combined extractswashed with saturated NaHCO₃ (30 ml), followed by saturated brine. Theorganic extract was then dried over MgSO₄. Removal of solvent andchromatography of the residue on Si gel with EtOAc-hexabes afforded357.3 mg (34%) of the title compound as a dark yellow-orange gum; ¹H NMR(CDCl₃) δ 0.79 (ddd, 1H), 1.16 (ddd, 1H), 1.26 (ddd, 1H), 1.38 (s, 3H),1.41 (ddd, 1H), 2.20 (s, 3H), 3.90 (s, 1H), 4.63 (s, 2H), 4.74 (s, 2H),7.25 (s, 1H), MS m/e 262 (M⁺); UV λ max (EtOH) 330.0 nm (ε 5011).

Example 3 Compound 3 (Formula I Wherein R₁ is Hydrogen; R₂ isAcetoxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0120] To a stirred solution of compound 1 (76.0 mg, 0.328 mmol) in 2 mlof acetic anhydride at room temperature, anhydrous sodium acetate (126.2mg, 1.54 mmol) was added. The mixture was stirred at room temperaturefor 24 hr, filtered to remove the NaOAc. The solution was placed underreduced pressure for 4 hr. The crude product was then partitionedbetween EtOAc and saturated NaHCO₃, followed by saturated brine, anddried over MgSO₄. After concentration the residue was chromatographed onSi gel with EtOAc-hexanes to give the title compound (50 mg, 56%) as ayellow gum; ¹H NMR (CDCl₃) δ 0.77 (ddd, 1H), 1.16 (ddd, 1H), 1.34 (ddd,1H), 1.38 (s, 3H), 1.58 (ddd, 1H), 2.06 (s, 3H), 2.11 (s, 3H), 4.99 (s,2H), 6.69 (s, 1H), 7.27 (s, 1H); ¹³C NMR (CDCl₃) δ 197.9, 170.8, 163.6,140.5, 140.0, 132.8, 126.7, 121.9, 76.7, 61.3, 37.9, 27.8, 20.9, 17.3,15.3, 10.3.

Example 4 Compound 4 (Formula I Wherein R₁ is Acetoxymethyl; R₂ isHydroxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0121] To a stirred solution of compound 10 (430 mg, 1.49 mmol) in 3 mlof EtOAc, 99% selenium dioxide (186 mg, 1.67 mmol) was added, followedby 90% tert-butyl hydroperoxide (0.40 ml, d 0.901, 4.0 mmol). Themixture was let stir at room temperature for 4 days, and thenpartitioned between EtOAc and saturated sodium sulfite (3×3 ml),followed by saturated brine, and dried over magnesium sulfate. Afterconcentration the crude product was chromatographed to give the titlecompound (8.5 mg, 2%) as a yellow gum; ¹H NMR (CDCl₃) δ 0.79 (ddd, 1H),1.17 (ddd, 1H), 1.39 (s, 3H), 1.43 (ddd, 1H), 1.52 (ddd, 1H), 2.09 (s,3H), 2.14 (s, 3H), 3.93 (s, 1H), 4.65 (q, 2H), 5.21 (q, 2H), 7.32 (s,1H); MS m/e 304 (M⁺); UV λ max (EtOH) 330.6 nm (ε 5950).

[0122] The intermediate compound 10 was prepared as follows.

[0123] a. Compound 10. Anhydrous sodium acetate was added to a stirredsolution of HMAF (1.4 g, 5.7 mmol) in acetic anhydride (6 mL) at roomtemperature. After 18 hours, the mixture was filtered and the resultingsolution was placed under reduced pressure for 4 hours. The resultingmaterial was partitioned between ethyl acetate and saturated aqueoussodium bicarbonate, followed by brine, and the resulting ethyl acetatesolution was dried over magnesium sulfate. The solution was concentratedand purified by chromatography on silica gel with ethyl acetate:hexanesas the eluent to give compound 10 (1.4 g, 85%) as a yellow-orange gum;¹H NMR (CDCl₃) δ 0.74 (ddd, 1H), 1.11 (ddd, 1H), 1.36 (s, 3H), 1.51(ddd, 1H), 2.04 (s, 3H), 2.09 (s, 3H), 2.17 (s, 3H), 3.90 (s, 1H), 5.10(s, 2h), 7.11 (s, 1H).

Example 5 Compound 5 (Formula I Wherein R₁ is Acetoxymethyl, R₂ isAcetoxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0124] To a stirred solution of compound 4 (2 mg, .0066 mmol) in 15 μlof CH₂Cl₂ and 135 μl of acetic anhydride, anhydrous NaOAc (3.1 mg, 0.038mmol) was added. The reaction was let stir at room temperature for 4 hr,filtered to remove the NaOAc. The mixture was placed under reducedpressure to remove the acetic anhydride. The crude product was thenpartitioned between EtOAc and saturated NaHCO₃, followed by saturatedbrine, and dried over MgSO₄. After concentration the residue waschromatographed on Si gel with EtOAc-hexanes to give the title compound(0.9 mg, 40%) as a yellow gum. ¹H NMR (CDCl₃) 6 0.79 (ddd, 1H), 1.15(ddd, 1H), 1.39 (s, 3H), 1.40 (ddd, 1H), 1.58 (ddd, 1H), 2.09 (s, 3H),2.093 (s, 3H), 2.10 (s, 3H), 3.89 (s, 1H), 5.08 (s, 2H), 5.17 (s, 2H),7.25 (s, 1H); MS m/e 346 (M⁺); UV λ max (EtOH) 332.1 nm (ε 8378).

Example 6 Compound 6 (Formula I Wherein R₁ is Hydroxymethyl; R₂ isAcetoxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0125] To a stirred solution of compound 2 (76.0 mg, 0.290mmol) in 1.5ml of acetic anhydride, anhydrous NaOAc (45.3 mg, 0.552 mmol) was added.The reaction mixture was stirred at room temperature for 75 min then,filtered to remove the NaOAc. The mixture was partitioned between EtOAcand saturated NaHCO₃, followed by saturated brine, and dried over MgSO₄.The extract was concentrated under reduced pressure and the residue waschromatographed on Si gel with EtOAc-hexanes to give compound 4 (6.9 mg)as a yellow gum, compound 2 (10.0 mg) as a yellow orange gum, 3 minorproducts including compound 5, and the title compound (10.0 mg, 11.3%)as a yellow gum; ¹H NMR (CDCl₃), 0.81 (ddd, 1H), 1.17 (ddd, 1H), 1.38(s, 3H), 1.60 (ddd, 1H), 2.06 (s, 3H), 2.24 (s, 31), 3.89 (s, 1H), 4.74(m, 2H), 5.03 (m, 2H), 7.23 (1H); MS m/e 304 (M⁺); UV λ max (EtOH) 331.3nm (ε 5921).

Example 7 Compound 7 (Formula I Wherein R₁ is Hydrogen; R₂ isMethoxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0126] To a stirred solution of compound 1 (300 mg, 1.29 mmol),in 10 mlof CH₃CN, 1 ml of CH₃I₂ and Ag₂O (110 mg, 0.475 mmol) were added. Themixture was stirred at room temperature for 3 days, then filtered andconcentrated. The residue was chromatographed on Si gel withEtOAc-hexanes giving compound 1 (133 mg) and the title compound (53 mg,17%) as a dark orange gum; ¹NMR (CDCl₃) δ 0.76 (ddd, 1H), 1.13 (ddd,1H), 1.34 (ddd, 1H), 1.38 (s, 3H), 2.05 (s, 3H), 3.41 (s, 3H), 3.96 (s,1H), 4.35 (s, 2H), 6.66 (s, 1H), 7.28 (s, 1H); ¹³C NMR (CDCl₃) 6 197.8,161.8, 143.3, 140.2, 133.1, 126.7, 120.9, 76.6, 69.6, 58.3, 37.6, 27.8,17.1, 14.9, 10.0.

Example 8. Compound 8 (Formula I Wherein R₁ is Methoxymethyl; R₂ isHydroxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0127] To a stirred solution of compound 11 (830 mg, 3.1 mmol) in 10 mlof EtOAc, 99% selenium dioxide (150 mg, 1.35 mmol) was added, followedby 90% tert-butyl hydroperoxide (1 ml, d 0.901, 10.0 mmol). The reactionmixture was stirred at room temperature for 5 days, partitioned betweendiethyl ether and saturated sodium sulfite, followed by saturated brine,and dried over MgSO₄. After concentration the crude material waschromatographed to give the title compound (47.2 mg, 5%) as a darkorange gum. ¹H NMR (CDCl₃) δ 0.74 (ddd, 1H), 1.08 (ddd, 1H), 1.32 (s,3H), 1.33 (ddd, 1H), 1.47 (ddd, 1H), 2.08 (s, 3H), 3.14 (br s, 1H), 3.35(s, 3H), 4.41 (q, 2H), 4.51 (br s, 2H), 7.28 (s, 1H).

[0128] a. Compound 11. The intermediate compound 11 (formula I whereinR₁ is methoxymethyl; R₂ is methyl; R₃ is hydrogen; R₄ is methyl; R₅ ishydroxy; and & is methyl) was prepared as follows.

[0129] To a stirred solution of HMAF (320 mg, 1.3 mmol) in 3 ml Me₂CO,MeOH (3 ml), and dilute H₂SO₄ (1M, 3 ml) were added. The reactionmixture was stirred at room temperature for 24 hours and extracted withdiethyl ether. The combined extracts were washed (saturated NaHCO₃,followed by brine), and dried over MgSO₄. After concentration theresidue was chromatographed on Si gel with EtOAc-hexanes affordingcompound 50 (290 mg, 86%) as a dark orange gum; 1H NMR (CDCl₃) 6 0.62(ddd, 1H), 0.98 (ddd, 1H), 1.24 (ddd, 1H), 1.27 (s, 3H), 1.37 (ddd, 1H),2.00 (s, 3H), 2.04 (s, 3H), 3.26 (s, 3H), 3.91 (br s, 1H), 4.29 (q, 2H),7.0 (s, 1H); ¹³C NMR (CDCl₃) δ 197.6, 159.7, 142.6, 138.8, 134.3, 129.7,126.6, 75.9, 65.3, 57.3, 37.3, 27.3, 15.7, 13.9, 12.9, 9.1.

Example 9 Compound 9 (Formula I Wherein R₁ is Methoxymethyl; R₂ isMethoxymethyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0130] To a stirred solution of compound 8 (240 mg, 0.87 mmol) in 15 mlof CH₃CN, 1.5 ml CH₃I, and Ag₂O (150 mg, 0.647 mmol) were added. Thereaction mixture was stirred at room temperature for 48 hr, filtered anconcentrated. The residue was chromatographed on Si gel withEtOAc-hexanes to give the title compound (25 mg, 10%) as a yellow-orangegum; ¹H NMR (CDCl₃) δ 0.76 (ddd, 1H), 1.11 (ddd, 1H), 1.37 (s, 3H), 1.40(ddd, 1H), 1.53 (ddd, 1H), 2.13 (s, 3H), 3.36 (s, 3H), 3.38 (s, 3H),3.91 (s, 1H), 4.37 (q, 2H), 4.43 (q, 2H), 7.27 (s, 1H), ¹³C NMR (CDCl₃)δ 198.0, 163.4, 142.8, 139.1, 132.6, 132.0, 127.2, 76.6, 67.2,65.5,58.3, 57.8, 38.1, 27.5, 16.3, 14.8, 9.8.

Example 10 Compound 12 (Formula I Wherein R₁ is—CH₂-[sulfur-linked-cysteine]; R₂is Methyl; R₃ is Hydrogen; R₄ isMethyl; R₅ is Hydroxy; and R₆ is Methyl)

[0131] To a solution of HMAF in acetone and 1M H₂SO₄ (1:1) was added oneequivalent of cysteine. The mixture was stirred at room temperatureovernight. A large amount of EtOAc was added and the water was removedby adding MgSO₄. Solid NaHCO₃ was also added in order to neutralize thesulfuric acid. The solution was filtered, concentrated, andchromatographed, to give compound 102.as a yellow gum: ¹H NMR (CDCl₃) δ0.78 (m, 1H), 0.89 (m, 1H), 1.06 (m, 1H), 1.31 (s, 3H), 1.43 (m, 1H),2.15 (s, 3H), 2.21 (s, 3H), 2.914.02 (m, 8H), 7.04 (s, 1H).

Example 11 Compound 13 (Formula I Wherein R₁ is—CH₂-[sulfur-linked-N-acylcysteine]; R₂ is Methyl; R₃ is Hydrogen; R₄ isMethyl; R₅ is Hydroxy; and R₆ is Methyl).

[0132] To a solution of HMAF (36 mg, 0.146 mmol) in 1:1 1MH₂SO_(4/)acetone (3 mL) was added N-acetyl cysteine (22.4 mg, 0.137mmol) at room temperature. The mixture was stirred for 22 hours and wasextracted with ethyl acetate. The organic extracts were washed withsaturated NaHCO₃ and saline respectively. The solution was dried overMgSO₄. After concentration, the crude product was chromatographed(adding 2-5% acetic acid to the normal solvent mixture, ethyl acetateand hexanes) to give 45.5 mg of compound 103 (85% yield) as a yellowgum; ¹H NMR (CDCl₃) δ 0.72 (m, 1H), 1.09 (m, 1H), 1.23 (m, 1H), 1.36 (s,3H), 1.47 (m, 11H), 2.07 (s, 3H), 2.10 (s, 3H), 2.13 (s, 31), 2.97 (m,1H), 3.14 (m, 1H), 3.82 (dd, 3.82), 4.80 (m, 211), 6.56 (d, J=7.2 Hz),7.10 (s, 1H); MS m/e 391 (M⁺), 373, 229, 185; HRMS for C₂₀H₂₅NO₅S calcd391.1455, found 391.1452.

Example 12 Compound 14 (Formula I Wherein R₁ is(S)-2-[(R)-x-methylbenzyl-aminocarbonyl]-2acylamino)ethylthiomethyl; R₂is Methyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0133] To a solution of compound 13 (40 mg, 0.102 mmol) in methylenechloride (I mid) was added N-hydroxybenzotriazole (20 mg, 0.132 mmol),N, N-diisopropylcarbodiimide (20 μL, 0.12 mmol) and (d)-(+)-α-methylbenzylamine (12 μL, 0.093 mmol). The mixture was stirred for 1.5 hoursat room temperature. The mixture was partitioned between EtOAc andwater. The organic extract was dried over MgSO₄. After concentration thecrude product was chromatographed to give 33.6 mg of compound 14 (73%)as a yellow gum; ¹H NMR (CDCl₃) δ 0.70 (m, 1H), 1.07 (m, 1H), 1.29 (m,1H), 1.35 (s, 3H), 1.48 (d, J=6.9 Hz, 3H), 1.51 (m, 1H), 1.93 (s, 3H),2.09 (s, 3H), 2.10 (s, 3H), 2.91 (m, 2H), 3.84 (dd, 2H), 4.61 (m, 1H),5.03 (m, 1H), 6.64 (d, J=7.8 Hz, 1H), 7.07 (s, 1H), 7.26 (m, 5H); ¹³CNMR (CDCl₃) δ 197.5, 170.4, 169.2, 159.7, 142.6, 142.0, 138.3, 134.9,129.8, 128.7, 127.4, 126.4, 125.9, 77.4, 52.5, 49.3, 37.7, 34.4, 29.8,27.6, 23.1, 22.1, 16.3, 14.3, 13.1, 9.5; HRMS for C₂₈H₃₄N₂O₄S calcd494.2241, found 494.2225.

Example 13 Compound 15 (Formula I Wherein R₁ is(R)-2-[(R)-α-methylbenzyl-aminocarbonyl]-2acylamino)ethylthiomethyl; R₂is Methyl; R₃ is Hydrogen; R₄ is Methyl; R₅ is Hydroxy; and R₆ isMethyl)

[0134] The chromatography from Example 12 also provided 5.3 mg ofcompound 15 (13%) as a yellow gum; ¹H NMR (CDCl₃) δ 0.70 (m, 1H), 1.07(m, 1H), 1.32 (m, 1H), 1.34 (s, 3H), 1.45 (m, 1H), 1.48 (d, J =6.9 Hz,3H), 2.03 (s, 3H), 2.05 (s, 6H), 2.76 (m, 1 )2.87 (m, 1), 3.73 (dd, 2H),4.50 (m, 1H), 5.03 (m, 1H), 6.46 (d, J=7.5 Hz, 1H), 6.77 (d, J=7.8 Hz,1H), 7.05 (s, 1H), 7.31 (m, 5H); HRMS for C₂₈H₃₄N₂O₄S calcd 494.2241,found 494.2238.

Example 14 Compound 16 (Formula I Wherein R₁ is—CH₂-[sulfur-linked-glutathione]; R₂ is Methyl; R₃ is Hydrogen; R₄ isMethyl; R₅ is Hydroxy; and R₆ is Methyl)

[0135] Using a procedure similar to that described in Example 10 exceptreplacing the cysteine used therein with glutathione, compound 16 wasprepared.

Example 15 General Procedure for the Synthesis of Compounds Wherein R₁is —CH₂-[sulfur-linked-cysteine]-peptide,—CH₂-[sulfur-linked-cysteine]-(Amino Acid),—CH₂-[sulfur-linked-N-acylcysteine]-peptide, or—CH₂-[sulfur-linked-N-acylcysteine]-(Amino Acid).

[0136] Using solid phase peptide synthesis techniques, which are wellknown in the art, the amino acids Leu, Phe and Gly were combined to givethe following resin bound tripeptides and dipeptides. -Leu-Leu-Leu-Leu-Leu-Phe -Leu-Leu-Gly -Leu-Phe-Leu -Leu-Phe-Phe -Leu-Phe-Gly-Leu-Gly-Leu -Leu-Gly-Phe -Leu-Gly-Gly -Phe-Leu-Leu -Leu-Leu-Phe-Phe-Leu-Gly -Phe-Phe-Leu -Phe-Phe-Phe -Phe-Phe-Gly -Phe-Gly-Leu-Phe-Gly-Phe -Phe-Gly-Gly -Gly-Leu-Leu -Gly-Leu-Phe -Gly-Leu-Gly-Gly-Phe-Leu -Gly-Phe-Phe -Gly-Phe-Gly -Gly-Gly-Leu -Gly-Gly-Phe-Gly-Gly-Gly -Leu-Leu -Leu-Phe -Leu-Gly -Phe-Leu -Phe-Phe -Phe-Gly-Gly-Leu -Gly-Phe -Gly-Gly

[0137] A rink acid resin having the following structure, wherein “P”represents a polystyrene divinylbenzene resin, was used in the solidphase reactions:

[0138] The first N-(9-fluorenylmethoxycarbonyl)-protected amino acid wascoupled to the resin via a bond formation between its C-terminus and thehydroxyl group of the resin using DEPC (Diisopropylcarbodiimide)/DMAP(4-Dimethylaminopyridine). The percent yield was calculated after thecoupling, and if it was below 90%, the coupling was repeated until thepercent yield exceeded 90%. The N-(9-fluorenylmethoxycarbonyl)-group(Fmoc-group) of the first amino acid was removed by treatment with 20%piperidine in 1-Methyl-2-Pyrrolidinone (NMP). A Kaiser's Test wasperformed to check the result of the deprotection, The three Kaiser'sreagents (Ninhydrin, Phenol and Potassium Cyanide) were added to analiquot of resin sample to yield a light yellowish solution. The mixturewas heated at 100° C. for 3 minutes, and if the solution turned darkpurple (positive result), the Fmoc-group was considered to have beenremoved. The next amino acid was coupled by peptide bond formation usingDIPC/HOBT(1-Hydroxy-Benzotriazole). NMP was used as the solvent fordeprotection coupling, and washing and, dichloromethane was used as thedrying reagent. Another Kaiser's test was then performed, with no colorchange (negative result) indicating that the coupling was successful.The coupling reaction was repeated to add additional amino acids.Compound 13 was coupled to the final resin bound amino acid or peptideusing dichloromethane as the solvent. NMP was not used as the solventfor the coupling reaction with compound 13, because NMP has been shownto cause inversion of the chiral center in cysteine, yielding a racemicmixture. Additionally, HOBT was not used in the coupling reaction withcompound 13, because it may also contribute to the formation of amixture of products. The cleavage of the resin from the peptide wasachieved by treatment with 10% acetic acid in dichloromethane. Theproducts were tested by UV and Mass Spectroscopy. HMAF gives two maximalUV absorbances at about 210 nm and 330 um, so the presence of productswas confirmed by the presence of these two absorbances. MassSpectroscopy was also used to confirm the presence of the desiredproducts.

[0139] Using this general procedure, compound 13 was coupled with the 36tri- and di-peptides shown above, and with the individual amino acidsLeu, Phe and Gly, to give a total of 39 compounds of the invention. TheN-acetyl group of these 39 compounds can also be removed usingconditions similar to those described in Example 11 to give anadditional 39 compounds of the invention. Accordingly, in a compound offormula I, R_(x) may preferably represent Leu, Phe, Gly, or any of the36 tri- and di-peptides shown above.

Example 16 The Following Illustrate Representative Pharmaceutical DosageForms, Containing a Compound of Formula I (‘Compound X’), forTherapeutic or Prophylactic Use in Humans

[0140] (I) Tablet I mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5Magnesium stearate 3.0 300.0 (ii) Tablet 2 mg/tablet ‘Compound X’ 20.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 500.0 (iii) Capsule mg/capsule ‘Compound X’10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinized starch120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg/ml) mg/ml‘Compound X’ (free acid form) 1.0 Dibasic sodium phosphate 12.0Monobasic sodium 0.7 phosphate Sodium chloride 4.5 1.0 N Sodiumhydroxide q.s. solution (pH adjustment to 7.0-7.5) Water for injectionq.s. ad 1 mL (v) Injection 2(10 mg/ml) mg/ml ‘Compound X’ (free acidform) 10.0 Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1Polyethylene glycol 400 200.0 01 N Sodium hydroxide solution q.s. (pHadjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (vi) Aerosolmg/can ‘Compound X’ 20.0 Oleic acid 10.0 Trichloromonofluoromethane5,000.0 Dichlorodifluoromethane 10,000.0 Dichlorotetrafluoroethane5,000.0

[0141] The above formulations may be obtained by conventional procedureswell known in the pharmaceutical art.

[0142] All publications, patents, and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound of formula I:

wherein R₁ is hydrogen, hydroxy, mercapto, amino, halo, carboxy, nitro,or —(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio, —N(R_(a))—, orabsent; Y is (C₃-C₆)cycloalkyl, aryl, heteroaryl, a saccharide, an aminoacid, a peptide, or a 1 to 15 membered branched or unbranched carbonchain optionally comprising 1, 2, or 3 non-peroxide oxy, thio, or—N(R_(a))—; wherein said chain may optionally be substituted on carbonwith 1, 2, or 3, oxo, hydroxy, carboxy, halo, mercapto, nitro,—N(R_(b))(R_(c)), (C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides,amino acids, or peptides; and wherein said chain may optionally besaturated or unsaturated; R₂ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, halo(C₁-C₆)alkyl, —C(═O)NR_(d)R_(e), asaccharide, an amino acid, a peptide, or (C₁-C₆)alkyl substituted by 1or 2 hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids,peptides, saccharides, or —C(O)NR_(d)R_(e); R₃ is hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl, heteroaryl,aryloxy, or heteroaryloxy; R₄ is hydrogen or (C₁-C₆)alkyl; and R₅ ishydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; or R₄ and R₅ takentogether are ethylenedioxy; R₆ is hydrogen, carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), asaccharide, an amino acid, a peptide, or (C₁-C₆)alkyl optionallysubstituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy,carboxy, amino acids, peptides, saccharides, or —C(═O)NR_(f)R_(g); R_(a)is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; and R_(b),R_(c), R_(d), R_(e), R_(f), and R_(g) are each independently hydrogen,(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) and R_(c)R_(d) and R_(e), or R_(f) and R_(g). together with the nitrogen to whichthey are attached, are pyrrolidino, piperidino, or morpholino; whereinany aryl, heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ mayoptionally be substituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl,hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto,nitro, or -N(R_(h))(R_(j)); wherein each R_(h) and R_(j) isindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl;or R_(h) and R_(j) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, or morpholino; or a pharmaceuticallyacceptable salt thereof.
 2. A compound of formula I:

wherein R₁ is —(CH₂)_(n)—(X)—(Y); n is 0to 4; X is oxy, thio, —N(R_(a))—or absent; Y is a monoprotected amino acid, a diprotected amino acid, apeptide, or a 1 to 15 membered branched or unbranched carbon chainoptionally comprising 1, 2, or 3 non-peroxide oxy, thio, or —N(R_(a))—;wherein said chain is substituted with 1, 2, or 3 peptides;/and whereinsaid chain may optionally be saturated or unsaturated; R₂ is hydrogen or(C₁-C₆)alkyl; R₃ is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; and R₅ is hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkanoyloxy; or R₄ and R₅ taken together are ethylenedioxy; R₆ ishydrogen, carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl,halo(C₁-C₆)alkyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl optionally substituted by 1 or 2 hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides,saccharides, or —C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; and R_(b), R_(c), R_(d), R_(e), R_(f)and R_(g) are each independently hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkanoyl, phenyl or benzyl; or R_(b) and R_(c), R_(d) and R_(e),or R_(f) and R_(g), together with the nitrogen to which they areattached, are pyrrolidino, piperidino, or morpholino; wherein any aryl,heteroaryl, aryloxy, or heteroaryloxy of Y, or R₃ may optionally besubstituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl,halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto, nitro, or-N(R_(h))(R_(j)); wherein each R_(h) and R_(j) is independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl or benzyl; or R_(h) andR_(j) together with the nitrogen to which they are attached arepyrrolidino, piperidino, or morpholino; or a pharmaceutically acceptablesalt thereof.
 3. A compound of formula I:

wherein R₁ is hydrogen, hydroxy, mercapto, amino, halo, carboxy, nitro,or —(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio, —N(R_(a))—, orabsent; Y is (C₃-C₆)cycloalkyl, aryl, heteroaryl, a saccharide, an aminoacid, a peptide, or a 1 to 15 membered branched or unbranched carbonchain optionally comprising 1, 2, or 3 non-peroxide oxy, thio, or—N(R_(a))—; wherein said chain may optionally be substituted on carbonwith 1, 2, or 3, oxo, hydroxy, carboxy, halo, mercapto, nitro,—N(R_(b))(R_(c)), (C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides,amino acids, or peptides; and wherein said chain may optionally besaturated or unsaturated; R₂ is hydrogen or (C₁-C₆)alkyl; R₃ ishydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkylthio, aryl,heteroaryl, aryloxy, or heteroaryloxy; R₄ is hydrogen or (C₁-C₆)alkyl;and R₅ is hydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; or R₄ and R₅taken together are ethylenedioxy; R₆ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, —C(═O)NR_(f)R_(g), a saccharide, an amino acid, apeptide, or (C₁-C₆)alkyl substituted by 1 or 2 (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(f)R_(g); R_(a) is hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; and R_(b), R_(c), R_(d), R_(e), R_(f) and R_(g) areeach independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl orbenzyl; or R_(b) and R_(c), R_(d) and R_(e), or R_(f) and R_(g) togetherwith the nitrogen to which they are attached, are pyrrolidino,piperidino, or morpholino; wherein any aryl, heteroaryl, aryloxy, orheteroaryloxy of Y, or R₃ may optionally be substituted by 1, 2, or 3(C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl, halo(C₁-C₆)alkyl, hydroxy,halo, carboxy, mercapto, nitro, or -N(R_(h))(R_(j)); wherein each R_(h)and R_(j) is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl,phenyl or benzyl; or R_(h) and R_(j) together with the nitrogen to whichthey are attached are pyrrolidino, piperidino, or morpholino; or apharmaceutically acceptable salt thereof.
 4. The compound of claim 1, 2,or 3 wherein R₁ is —(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio,—N(R_(a))—, or absent; and Y is a peptide, or (C₁-C₆)alkyl substitutedwith a peptide.
 5. The compound of claim 1 or 3 wherein R₁ is hydroxy,halo, carboxy, aryl; heteroaryl, a saccharide, an amino acid, or apeptide.
 6. The compound of claim 1 or 3 wherein R₁ is hydrogen or(C₁-C₆)alkyl, optionally substituted with 1 or 2 hydroxy, halo, methoxyor ethoxy.
 7. The compound of claim 1 or 3 wherein R₁ is—(CH₂)_(n)—(X)—Y); n is 1 or 2; X is oxy, thio, or —N(R_(a))—; and Y is(C₁-C₆)alkyl or (C₂-C₆)alkenyl, optionally substituted with 1 or 2 oxo,hydroxy, carboxy, halo, mercapto, nitro, —N(R_(b))(R_(c)),(C₃-C₆)cycloalkyl, aryl, heteroaryl, saccharides, amino acids, orpeptides; wherein any aryl or heteroaryl of Y may optionally besubstituted by 1, 2, or 3 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkanoyloxy, (C₁-C₆)alkoxycarbonyl, hydroxy(C₁-C₆)alkyl,halo(C₁-C₆)alkyl, hydroxy, halo, carboxy, mercapto, nitro, and—N(R_(h))(R_(j)); wherein each R_(h) and R_(j) is independentlyhydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkanoyl, phenyl and benzyl; or R_(h) andR_(j) together with the nitrogen to which they are attached form apyrrolidino, piperidino, or morpholino radical.
 8. The compound of claim1, 2, or 3 wherein R₁ is —CH₂-[sulfur-linked-cysteine]-R_(x) or—CH₂-[sulfur-linked -N-acylcysteine]-R_(x), wherein R_(x) is an aminoacid or a peptide comprising 2 to 24 amino acids.
 9. The compound ofclaim 8 wherein F, is -Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO:4),-Asp-Gly-Arg-Cys (SEQ ID NO:5), or-Asp-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO:6).10. The compound of claim 8 wherein R_(x) is a peptide consisting of 4to 20 amino acids.
 11. The compound of claim 1 or 3 wherein R₁ is—CH₂-[sulfur-linked-cysteine] or —CH₂-[sulfur-linked-N-acylcysteine].12. The compound of claim 1, 2, or 3 wherein R₁ is2-[(R)-α-methylbenzylaminocarbonyl]-2-(acylamino)ethylthiomethyl. 13.The compound of claim 1, 2, or 3 wherein R₁ is—CH₂-[sulfur-linked-glutathione].
 14. The compound of claim 1 or 3wherein R₁ is hydrogen, methyl, hydroxymethyl, methoxymethyl, oracetoxymethyl.
 15. The compound of claim 1 wherein R₂ is hydroxymethyl,methoxymethyl, or acetoxymethyl.
 16. The compound of claim 1 wherein R₂is carboxy, (C₁-C₆)alkanoyl, (C₁-C₆)alkoxycarbonyl, or—C(═O)NR_(d)R_(e).
 17. The compound of claim 1 wherein R₂ is(C₁-C₆)alkyl substituted by 1 or 2 hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, carboxy, amino acids, peptides, saccharides, or—C(═O)NR_(d),R_(e).
 18. The compound of claim 1, 2, or 3 wherein R₄ ismethyl.
 19. The compound of claim 1, 2, or 3 wherein R₅ is hydroxy. 20.The compound of claim 1 or 2 wherein R₆ is hydrogen.
 21. The compound ofclaim 1, 2, or 3 wherein R₆ is carboxy, (C₁-C₆)alkanoyl,(C₁-C₆)alkoxycarbonyl, or —C(═O)NR_(f)R_(g).
 22. The compound of claim 1or 2 wherein R₆ is (C₁-C₆)alkyl optionally substituted by 1 or 2hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy, carboxy, amino acids,peptides, saccharides, or —C(═O)NR_(f)R_(g).
 23. The compound of claim 1or 2 wherein R₆ is methyl or hydroxymethyl.
 24. The compound of claim 1wherein R₂ is (C₁-C₆)alkyl, substituted with hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy, or carboxy; R₃ is H, (C₁-C₆)alkyl, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, aryl, heteroaryl, aryloxy, or heteroaryloxy; R₄ ishydrogen or (C₁-C₆)alkyl; R₅ is hydroxy; and R₆ is (C₁-C₆)alkyl,optionally substituted with hydroxy, (C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy,or carboxy; or a pharmaceutically acceptable salt thereof.
 25. Thecompound of claim 1 wherein R₁ is hydrogen or (C₁-C₆)alkyl, optionallysubstituted with hydroxy, halo, methoxy, ethoxy, or acetoxy; R₂ ishydroxymethyl, methoxymethyl, or acetoxymethyl; R₃ is hydrogen; R₄ ismethyl; R₅ is hydroxy; and R₆ is methyl or hydroxymethyl; or apharmaceutically acceptable salt thereof.
 26. The compound of claim 2wherein R₁ is —(CH₂)_(n)—(X)—(Y); n is 0 to 4; X is oxy, thio,—N(R_(a))—, or absent; and Y is a monoprotected amino acid or adiprotected amino acid.
 27. The compound of claim 2 wherein R₁ is—CH₂-[sulfur-linked-N-acylcysteine],(S)-2-[(R)-α-methylbenzylaminocarbonyl]-2-(acylamino)ethyl-thiomethyl,or(R)-2-[(R)-α-methylbenzylaminocarbonyl]-2-(acylamino)ethyl-thiomethyl.28. A dimeric compound comprising two compounds of claim 1 connected bya linker.
 29. A method comprising inhibiting cancer cells, by contactingsaid cells, in vitro or in vivo, with an effective amount of a compoundof claim 1, 2, or
 3. 30. A therapeutic method Comprising treating cancerby administering an effective amount of a compound of claim 1, 2, or 3to a mammal in need of such therapy.
 31. A compound comprising acompound of claim 1, 2, or 3, and a reagent that is capable of bindingto a tumor-associated antigen.
 32. A pharmaceutical compositioncomprising one or more compounds of claim 1, 2, or 3 in combination witha pharmaceutically acceptable diluent or carrier.